JPS6121084A - Bacteria counter - Google Patents

Abstract

PURPOSE:To detect the amount of bacteria in water to be measured and reduce the production cost of semiconductors, etc., by passing specific water to be measured through a measurement chamber with a small exciting light irradiation volume, irradiating the water with exciting light to measure the emitted fluorescence. CONSTITUTION:Ultrapure water as water to be measured is fed from a line 21 through a branch pipeline 22 to a reaction chamber 23, and acridine orange (derivative) is added, reacted with bacteria therein, pressurized with N2 gas fed through a valve 25 and then fed into a measurement chamber 28 having an exciting light irradiation part with a reduced thickness and irradiation area in the exciting light irradiation direction. On the other hand, excited light at 440- 500nm, e.g. argon laser, 488nm, is irradiated from an exciting light source 29 through a condenser lens 30 and galvanomirror 31 in the direction perpendicular to the passing direction of the water to be measured to emit fluorescence, which is then inputted through a photomultiplier 34, amplifier 35 and differentiation circuit 37 to a pulse counter 38 to count the number of the bacteria.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to a bacterial counter, and particularly to a bacterial counter for measuring the amount of bacteria in ultrapure water used in semiconductor manufacturing, etc. be.

In water quality testing, in addition to testing the types of bacteria contained in the sample water, it is important to test how many bacteria are present per unit amount of water. .

This inspection of the amount of bacteria can be carried out, for example, in cases where only an extremely small amount of bacteria is allowed in the manufacture of pharmaceuticals, or in the manufacture of semiconductors (particularly ultra-LSI semiconductors), where only an amount of about 10 or less bacteria is allowed per occc. It can be said that this test is indispensable in fields where ultrapure water is required, such as in cases where ultrapure water is not used.

Conventionally, the method for testing the amount of bacteria per unit amount of water is to collect manufactured ultrapure water, culture and multiply the bacteria contained in it, and then dye the filtered bacteria. A common method is to then observe and count the amount of stained bacteria using a microscope.

However, using this type of testing method, it takes about a week from collecting ultrapure water to counting the amount of bacteria, so even if the water quality is found to be unsuitable by this test, the product cannot be manufactured during this testing period. There is a great possibility that semiconductor devices and the like that have been used will become defective products, resulting in an increase in production costs.

Therefore, the present inventors first proposed a bacteria counter that can detect the amount of bacteria in the measurement water in an extremely short period of time, and can reduce the production cost of semiconductors etc. by constantly monitoring the amount of bacteria. did.

The bacteria counter proposed by the present inventors consists of a translucent measurement chamber, into which measurement water to which acridine orange or its derivatives have been added and which has generated a reaction product with the bacteria contained therein is introduced. An excitation light source for irradiating the measurement water with excitation light to generate fluorescence from the reaction product, a photoelectric converter for receiving the fluorescence, and converting the output of this photoelectric converter into fluorescence output per unit bacteria. The basic configuration is to have an arithmetic unit for outputting.

FIG. 1 is a schematic diagram showing the main parts of a bacteria counter based on this basic configuration. Reference numeral 1 denotes a reaction chamber in which acridine orange or a derivative thereof (for example, 3-amino-6-medoxyacridine, etc.) is added to and reacted with measurement water supplied from a pure water line led to semiconductor manufacturing equipment.

The measurement water in which reaction products have been produced in the reaction chamber 3 is then led to the measurement chamber 2. This measurement chamber 2 is made of a transparent material such as quartz, and in this example, the space (inner wall space) into which the measurement water is guided is 10crn x 10crn.
It is formed into a square.

3 is an excitation light source, and in this example, a 2.5 mW argon laser (wavelength 488 nm) is used. A lens 4 irradiates the measurement water in the measurement chamber 2 with the excitation light from the light source 3 as a beam of about 0.5φ. Reference numerals 5, 6, and 7 denote an interference filter, a condenser lens, and a photomultiplier for receiving fluorescence generated by irradiating the reaction product in the measurement water with excitation light and generating an output.

The output of the photomultiplier 7, which has received fluorescence corresponding to the amount of reaction products in the measurement chamber 2, is input to the computing unit via the Rotstein amplifier. This calculator is for calculating the amount of bacteria by converting the output of the photomultiplier 7, which corresponds to the amount of bacteria in the measurement water, into the output per unit of bacteria. By converting the amplitude value of the output from 7 into the amplitude value per unit bacteria, the amount of bacteria contained in the measurement water can be calculated.

<Problems to be Solved by the Invention> Using the apparatus shown in Figure 1, the number of cuticles was varied in measurement water to which 50 μf of acridine orange was added per 100 cc of pure water, and photomultiglare counting was performed. The value was measured. The results are shown in Table 1 and Figure 2A.

Similarly, for the purpose of investigating the thermal aging of acridine orange, the photomultiplier was measured when a 5 mW argon laser was continuously irradiated to measurement water (no bacteria added) in which 20 μt of acridine orange was added per 1000 C of pure water. Count values were measured.

The results are shown in Table 2 and Figure 2B.

Table 2 Furthermore, for the purpose of measuring the rate of change of the photomultiplier and laser, the count value of the photomultiplier was measured in the same manner as described above using a sample water consisting of 100 cc of pure water. The results are shown in Table 3 and Figure 2C.

As is clear from Table 3, Figure 2A, the detection limit for the amount of bacteria in the above device is 800 per cc of measured water based on the fluorescence noise shown in the symbol and the variation in each count value.
The number is about 1,000 to 1,000. Therefore, this device is not sufficient for tests that require detection of 10 or fewer bacteria per animal, which is necessary for the manufacture of semiconductor devices. Fluorescence noise, which is the biggest factor in the above detection limit, is caused not only by acridine orange that reacts with bacteria and generates fluorescence when measurement water is irradiated with excitation light, but also by free acridine orange present in pure water. It has also become clear that this is also based on the emission of fluorescence.

<Objective of the Invention> The present invention provides a bacteria counter that can detect the amount of bacteria in measurement water in an extremely short time and can reduce the production cost of semiconductors and the like by constantly monitoring the amount of bacteria. The purpose of this invention is to provide a bacteria counter capable of detecting even a very small amount of bacteria required for semiconductor manufacturing.

Configuration for Solving the Problems> The structure of the bacteria counter of the present invention for solving the above problems is as follows: A light-transmitting measurement chamber into which pure water to be measured is introduced; an excitation light source for irradiating the measurement water in the measurement chamber with excitation light to generate fluorescence from the reaction product; The thickness of the measurement chamber space in the excitation light irradiation direction and the irradiation area of the excitation light are reduced so that the fluorescence from the reaction product in the excitation light irradiation section of the measurement chamber is transmitted to the reagent. The excitation light irradiation volume is made small so that the excitation light irradiation volume is sufficiently larger than that of a single fluorescence, and a pressurizing means is provided for causing measurement water to flow into the measurement chamber.

<Example> Hereinafter, the present invention will be explained in more detail with reference to the drawings.

First, regarding the causes of the bacteria measurement limit in the apparatus shown in Fig. 1, we will discuss the fluorescence intensity per bacterium (assumed to be 1 μm in size) B and the fluorescence intensity IRA per 11i of pure water containing acridine orange ( Acridine orange ni concentration). Here, the measurement chamber space was 10 m x 10 crn, and the laser beam size was 0.5 rns x 0, 5 u (see Fig. 3).

Number of bacteria present in the irradiated area: 2.5 x 10-3・NB, -
(1) Here, NB is the number of bacteria per unit volume (cd), the volume of the irradiated area, 2.5 x 10' μ? - (2) Total fluorescence intensity measured i I = (1) X IB + (2) X I pigeon
-(3) Here, from the above experimental results, nil 180 IAQ-f-i 7.2 x 10-8 counts/1μ-1 (4) 4 counts/1μ---(5).

Formulas (4) and (5) correspond to acridine orange molecular weights of 105 and 1012, respectively.

On the other hand, since the total number of l unit cells in the laser irradiation section is 2.5 x 10' as shown in Figure M4, a simulation using the number of bacteria in the irradiation section as a parameter results in the results shown in Table 4.

As is clear from the above simulation results, by reducing the volume of the laser irradiation part, fluorescence noise can be lowered and the detection limit for the amount of bacteria can be increased. For example, the space in the measurement chamber in the direction in which the laser is irradiated is 100 μfF! Thickness, laser beam diameter 100
Assuming 1tfn, water (free) in the irradiated area
The number of acridine oranges is I x 1011, and the measurement solution in which 1 bacterium is present in 1 cC is also diN.
≧10, it becomes detectable.

The present invention will now be described in detail with reference to FIG. 21
23 is an ultrapure water line led to, for example, semiconductor manufacturing equipment, and 23 is a line of ultrapure water that is supplied from the ultrapure water line 21 through a branch pipe 22 to acridine orange or a derivative thereof (for example, 3-amino-6 -Medoxyacridine, etc.)
This is a reaction chamber in which the components are added and reacted. Acridine orange or its derivatives reacts with the bacterial DNA (dioxy, acid, which is the core of the protein) in the measurement water to produce a reaction product with high density in the bacteria, and this product is released by irradiation with excitation light. Generates relatively high fluorescence. For example, about 440 to 500 nm as excitation light
An argon laser (eg, 488 nm) is used to generate fluorescence at about 500 to 580 nm (peak value about 530 nm).

The N and gas supplied to the reaction chamber 23 are ultrapure water with acridine orange or a derivative thereof added to the measurement chamber 2.
This is the pressurized gas to be injected into 8. Further, 24, 25 and 26 are valves for ultrapure water, N, gas, and measurement water, respectively, and by adjusting these valves, the passage speed of measurement water in the measurement chamber 28 is controlled. Also, 27 is a valve for the addition of acridine orange reagent.

In the reaction chamber 23, the measurement water in which the reaction products were produced is N! The gas is injected into the measurement chamber 28 due to its pressure. As shown in the cross section of FIG. 5, this measurement chamber 28 forms a rectangular passage whose space has an extremely small thickness in the irradiation direction of the excitation light.

The thickness of the space in the measurement chamber 28 is determined by the ratio of the signal to S based on the volume of the excitation light irradiation section described above. for example,
This space is a space with a rectangular cross section of 100 to 200 μm x 10 fins. The measurement chamber 28 irradiates the measurement water in which the reaction product has been generated with excitation light to emit fluorescence,
The tube is used to measure the amount of fluorescence, and must be at least partially transparent to light in the wavelength range of the excitation light and fluorescence. For example, a quartz tube is used.

29 is an excitation light source, for example, an argon laser (488
nm) is used, a condensing lens 30, a galvanometer mirror
The excitation light is irradiated to the measurement water in the measurement chamber 28 via 31.

The condensing lens 30 is used to reduce the volume of the excitation light irradiation part of the measurement chamber 28 by focusing the excitation light to a diameter of about 10011fn, for example. In addition, the galvanometer mirror 31 is located in the measurement chamber 2.
8, the excitation surface is scanned in a direction perpendicular to the direction in which the measurement water passes through.

The relationship between the scanning speed of the excitation light by the galvanometer mirror 31 and the passing speed of the measurement water passing through the measurement chamber 28 can be determined, for example, as follows. For example, assuming that the space of the measurement chamber 28 has a thickness of 0.1 to 0.27 ui and a width of 8 [11],
If 10cc of measurement water is extruded and flowed, and the measurement time is 2 minutes, the flow rate is 0.083'/see.
Therefore, the linear velocity in the measurement chamber 28 is l at a thickness of 0.1io+
Q, 4 cm/8eo1 thickness 0.2-5.2a
It's 8o0. Since the laser diameter is 100 μm, in order to irradiate the entire sample water with excitation light, the scanning speed is 520 Hz for a thickness of 0.1 M and 260 Hz for a thickness of 0.2 m.
That would be a good thing.

A photomultiplier 34 receives fluorescence generated by irradiating the measurement water in the measurement chamber 28 with excitation light and generates an output based on the fluorescence intensity. 32 and 33 are an interference filter and a condensing optical system arranged on the incident side of the photomultiplier 34. The photomultiplier 34 is preferably disposed in a direction perpendicular to the optical axis of the excitation light in order to prevent noise from occurring due to the incidence of the excitation light.

A photomultiplier that receives fluorescence in this way
As shown in FIG. 6, the output of 34 is amplified by an amplifier 35 and then input to an oscilloscope, so that the occurrence of fluorescence can be detected.

Furthermore, the output of this amplifier can be inputted to a pulse counter 38 via a differentiating circuit 37 to directly count the number of bacteria. Reference numeral 39 is a drive source for driving the galvano mirror 31, oscilloscope 36, etc., and 40 is a drive source for the laser light source.

Figure 7 shows the spherical mirror 4 that guides the fluorescence generated by irradiating the excitation light onto the measurement water to the photomultiplier 34 at the opening rate.
1 is provided to surround the measurement chamber 28, and this spherical mirror 41 has openings 42 and 43 for inputting and outputting excitation light.
A photomultiplier 34 is further attached.

In the above embodiment, the amount of bacteria in the entire sample water passing through the measurement chamber was detected by scanning the flow of sample water and excitation light, but some bacteria in the sample water passing through the measurement chamber were detected. The number of bacteria may be detected and the total number of bacteria may be calculated based on this value. In this case, scanning of the excitation light is not necessarily required.

Furthermore, in the above embodiment, only the number of bacteria was detected, but when the excitation light is irradiated onto the measurement water in the measurement chamber, the reflected light in the measurement water (light with the same wavelength as the excitation light) ) can be further measured to detect the amount of impurities in the measurement water. This allows bacteria and non-bacterial impurities to be counted. The measurement of the excitation light reflection does not necessarily have to be carried out in a measurement chamber for detecting bacteria, and may be carried out by arranging two measurement chambers in series.

<Effects of the Invention> As explained in detail above, according to the present invention, the amount of bacteria in the measurement water can be detected in an extremely short time, and the production cost of semiconductors, etc. can be reduced by constantly monitoring the amount of bacteria. It becomes possible to do so. In particular, since it is possible to detect extremely small amounts of bacteria required for semiconductor manufacturing, etc., it is possible to have a significant effect on reducing semiconductor production costs.

[Brief explanation of drawings]

Fig. 1 is a perspective view showing the outline of the bacteria counter previously proposed by the present inventors; FIG. 4 is a conceptual diagram showing the state of the excitation light irradiation section, FIGS. 5 and 6 are a perspective view and a Tachibana diagram showing one embodiment of the present invention, and FIG. 7 is a diagram showing another embodiment of the present invention. FIG. In the figure, 21 is a pure water line, 23 is a reaction chamber, 28 is a measurement chamber, 29 is an excitation light source, 31 is a galvanometer mirror 1, and 34 is a photomultiplier.

Claims (5)

[Claims] (1) A translucent measurement chamber into which the measurement water, which is ultrapure water to which reagents such as acridine orange have been added to produce reaction products with the bacteria it contains, and the measurement water in this measurement chamber. It has an excitation light source for emitting excitation light to generate fluorescence from the reaction product, and a photoelectric converter for receiving the fluorescence, and the thickness of the measurement chamber space in the direction of excitation light irradiation and the excitation. The volume of excitation light irradiation is made small so that the fluorescence from the reaction product in the excitation light irradiation part of the measurement chamber is sufficiently larger than the fluorescence of the reagent alone, and the measurement water is placed in the measurement chamber. A bacteria counter comprising: a pressurizing means for causing an inflow of bacteria. (2) The bacteria counter according to claim 1, wherein the excitation light source is a laser light source. (3) The bacteria counter according to claim 1 or 2, further comprising a mechanism for scanning excitation light in a direction perpendicular to the inflow direction of the measurement water into the measurement chamber. (4) The volume of the excitation light irradiation part of the measurement chamber is 10c. c.
．． 2. The bacteria counter according to claim 1, wherein the bacteria counter is configured to be small enough to detect one bacterium per bacteria. (5) The bacteria counter according to claim 1, characterized by comprising a circuit for differentiating the output of a photoelectric converter for receiving fluorescence, and a counter for counting this output.